Localization of the FGR protooncogene on the genetic linkage map of human chromosome 1p

A restriction fragment length polymorphism (RFLP) at the human FGR gene, a member of the src family of protooncogenes, has been identified and used to locate FGR on the genetic linkage map of human chromosome 1p. Single-copy sequences subcloned from a cosmid containing the human FGR gene were used to screen a panel of genomic DNAs for RFLPs. One plasmid, designated pB8, detected a high-frequency EcoRI RFLP (allele frequencies, 0.57/0.43). Analysis of a panel of somatic cell hybrids demonstrated that pB8 maps to the region 1p31-pter. Genetic linkage analysis of the 40 families provided by the Centre d'Etude du Polymorphisme Humain (CEPH) showed that FGR maps to a location 3.1 cM from the Rh blood group locus (RH), and falls in the 17.5-cM gap between alpha-fucosidase (FUCA1) and D1S57. The relative gene order of RH and FGR could not be determined unequivocally, but the most favored gene order was 1pter-PND-ALPL-FUCA1-FGR-RH-D1S57-MYCL.     

Localization of a DNA segment encompassing four tRNA genes to human chromosome 14q11 and its use as an anchor locus for linkage analysis

The chromosomal location of an 8.2-kb genomic fragment encompassing a cluster of four human tRNA genes has been determined by three complementary methods including Southern analysis of human/rodent somatic cell hybrids, in situ hybridization, and genetic linkage analysis. This tRNA cluster (TRP1, TRP2, and TRL1) is located near the T-cell receptor alpha (TCRA) locus at 14q11, and several RFLPs were detected at this site. These RFLPs and those at the TCRA and MYH7 (cardiac beta-MHC gene) loci have been used to type all informative members of the CEPH pedigrees. This has permitted ordering of these three gene loci and two anonymous probes (D14S26 and D14S25) in a 20-cM interval just below the centromere of chromosome 14. Based upon the chromosomal location and the polymorphisms at this site, one or more members of this gene cluster could serve as a useful anchor locus on chromosome 14.     

Polymorphism of the T-cell receptor gamma variable and constant region genes in a Chinese population

Summary The human T-cell receptor gamma gene region spans 160 kb genomic DNA. Restriction fragment length polymorphisms (RFLPs) have been previously documented for the constant region (TRGC) genes, the joining (TRGJ) segments and the variable (TRGV) genes. We have recently defined the alleles of the T-cell receptor gamma V, J and C genes and we have described seven haplotypes of the V gamma subgroup I genes characterized either by RFLPs or by deletion or insertion of V gamma genes. The number of VI genes may vary from 7 to 10 per haploid genome, the 9-gene haplotype being the most frequent. Allelic fragments can unambiguously characterize the TRGC2 gene with duplication or triplication of the exon 2. These alleles and haplotypes have been analyzed in four different populations (French, Lebanese, Tunisian and Black African). In this paper, we compare these allele and haplotype frequencies with those found in a Chinese population and we describe new TRGV allelic restriction fragments found only in the Chinese samples. These results and the previous data demonstrate the flexibility of the human T cell receptor gamma locus and the importance of unequal crossing-overs in the evolution of that locus. Moreover, they underline the importance of studying these polymorphisms in population genetics.     

Polymorphic DNA haplotypes at the LDL receptor locus.

Mutations in the low-density lipoprotein (LDL) receptor gene result in the autosomal dominant disorder familial hypercholesterolemia (FH). Many different LDL receptor mutations have been identified and characterized, demonstrating a high degree of allelic heterogeneity at this locus. The ability to identify mutant LDL receptor genes for prenatal diagnosis of homozygous FH or to study the role of the LDL receptor gene in polygenic hypercholesterolemia requires the use of closely linked RFLPs. In the present study we used 10 different RFLPs, including three newly described polymorphisms, to construct 123 independent haplotypes from 20 Caucasian American pedigrees. Our sample contained 31 different haplotypes varying in frequency from 0.8% to 29.3%; the five most common haplotypes account for 67.5% of the sample. The heterozygosity and PIC of each site were determined, and these values disclosed that eight of the RFLPs were substantially polymorphic. Linkage-disequilibrium analysis of the haplotype data revealed strong nonrandom associations among all 10 RFLPs, especially among those sites clustered in the 3' region of the gene. Evolutionary analysis suggests the occurrence of both mutational and recombinational events in the generation of the observed haplotypes. A strategy for haplotype analysis of the LDL receptor gene in individuals of Caucasian American descent is presented.     

Characterization of the Mouse Rh Blood Group Gene

The Rh blood group system is of clinical importance in blood transfusion and as the cause of hemolytic disease of the newborn. Other than their role as carriers of Rh antigens, very little is known about the function of the Rh polypeptides. As a first step to generate an animal model system in which to study the structure and function of Rh, and to extend the phylogenetic analysis of RH genes, the Rh homologue from Mus musculus was characterized. Comparison of RH from humans and mice revealed 71 and 58% sequence identity at the nucleotide and amino acid levels, respectively. Mouse Rh mRNA encodes a protein which is 1 amino acid longer (418 aa) than that of human (417 aa). Rh protein was detected in mouse erythrocyte membranes and was comparable in size to human Rh. Mouse erythrocytes do not show serologic reactivity with human Rh antibodies, probably because the greatest divergence between the mouse and the human genes was seen in the predicted extracellular loops, while the transmembrane regions were more conserved. The mouse RH locus consists of only one gene, which is important for future genetic manipulation and which also indicates that the RH gene duplication seen in humans has occurred since the mammalian radiation.     

A high-resolution radiation hybrid map of the proximal portion of mouse chromosome 5

Radiation hybrid (RH) mapping of the mouse genome provides a useful tool in the integration of existing genetic and physical maps, as well as in the ongoing effort to generate a dense map of expressed sequence tags. To facilitate functional analysis of mouse Chromosome 5, we have constructed a high-resolution RH map spanning 75 cM of the chromosome. During the course of these studies, we have developed RHBase, an RH data management program that provides data storage and an interface to several RH mapping programs and databases. We have typed 95 markers on the T31 RH panel and generated an integrated map, pooling data from several sources. The integrated RH map ranges from the most proximal marker, D5Mit331 (Chromosome Committee offset, 3 cM), to D5Mit326, 74.5 cM distal on our genetic map (Chromosome Committee offset, 80 cM), and consists of 138 markers, including 89 simple sequence length polymorphic markers, 11 sequence-tagged sites generated from BAC end sequence, and 38 gene loci, and represents average coverage of approximately one locus per 0.5 cM with some regions more densely mapped. In addition to the RH mapping of markers and genes previously localized on mouse Chromosome 5, this RH map places the alpha-4 GABA(A) receptor subunit gene (Gabra4) in the central portion of the chromosome, in the vicinity of the cluster of three other GABA(A) receptor subunit genes (Gabrg1-Gabra2-Gabrb1). Our mapping effort has also defined a new cluster of four genes in the semaphorin gene family (Sema3a, Sema3c, Sema3d, and Sema3e) and the Wolfram syndrome gene (Wfs1) in this region of the chromosome.     

Identification and characterization of nine RFLPs at the adenosine deaminase (ADA) locus.

We have identified and/or characterized at least nine RFLPs at the adenosine deaminase (ADA) locus, detected by digestion of DNA with MspI, BanII, PstI, BalI, and PvuII. The RFLPs were distributed over approximately 15 kb of the gene, from IVS 2 to IVS 10. They exhibited Mendelian inheritance and were in Hardy-Weinberg equilibrium. For seven fully characterized RFLPs, the gene frequencies of the rare alleles in 90 chromosomes examined ranged from .33 to .04, the PIC from .34 to .07, and the heterozygosity from .09 to .58. In kindreds examined (58 independent chromosomes), a total of nine haplotypes could be defined on the basis of seven fully characterized RFLPs with a heterozygosity of .62 and PIC of .53. Because there was considerable linkage disequilibrium, only three haplotypes accounted for 90% of individuals. Similar heterozygosity and PIC values (.59 and .51, respectively) could be obtained on the basis of haplotypes defined by the two sites that were the most polymorphic and that were in the least degree of linkage disequilibrium. A strategy for use of the RFLPs in linkage studies is suggested. We have also examined DNA from 17 patients with complete genetic deficiency of ADA (resulting in severe combined immunodeficiency [ADA-SCID] and from 10 patients with partial ADA deficiency (deficient in erythrocytes, with varying levels of ADA in other cells and normal immune function). Although the RFLPs detected genetic compounds among both types of patients, there was, as expected, a decreased incidence of heterozygosity (ADA-SCIDs, .29; partial ADA deficients, .20). Two additional haplotypes not found in the normal population were identified in homozygous form in patients. This information should be useful in developing a rational approach to delineation of mutations at the ADA locus as well as in distinguishing recurrent mutations of independent origin from those derived from a common progenitor.     

Genetic structure of the Mongols as derived from the ABO, MN, Rh, EsD, GLO1, PGM1, AcP, 6-PGD, Hp, Gc, Tf, C'3 and ChE2 loci

According to integral characterization of gene frequencies of the investigated loci AB0, MN, Rh, GLO1, PGM1, EsD, AcP, 6-PGD, Hp, Tf, Gc, C'3 and ChE2, Mongolian population has high level of polymorphism, with the exception of haplotypes R" (cdE) and Ry(CdE) at the Rh locus and TfB0-1 at the Tf locus. The data on biochemical and immunological polymorphic gene markers analysed in the population of Mongolia show that the Mongolians have some distinctive features, in comparison with the mean-in-the-world characteristics: high frequencies of the B genes at the AB0 locus; D, E, R1 and R2 at the Rh locus; GLO11, PGDc, TfDChi, E2(C5+), PGM1(1+); low frequencies of the genes A(AB0), R0(Rh), AcPc, Hp1, Gc2, C'3F, PGM 1(2-); the rest of the genes at the above-mentioned loci and the genes of the locus MN have the mean-in-the-world frequencies.     

The ornithine aminotransferase (OAT) locus: analysis of RFLPs in gyrate atrophy.

A cDNA probe (HOAT1) for ornithine aminotransferase (OAT) has recently been used to map (1) the structural gene for this enzyme to chromosome 10 and (2) several related DNA sequences to the X chromosome. We have defined six RFLPs for OAT, to explore its possible role in gyrate atrophy (GA) of the choroid and retina, an autosomal recessive genetic disorder associated with a deficiency of OAT activity. The RFLPs, which are detected by noncoding single-copy probes from the OAT gene and by subclones of the HOAT1 cDNA, all map on human chromosome 10, producing an overall level of heterozygosity for the OAT locus of 83%. Using the RFLPs, we have determined that the OAT locus segregates concordantly with GA in one available pedigree. Furthermore, the RFLPs display significant disequilibrium with GA, providing genetic evidence implicating a defect in the OAT structural gene as the cause of this disorder. The RFLPs for OAT are potentially applicable to prenatal diagnosis and carrier detection in families with a previous history of GA. They will also allow identification of specific haplotypes associated with GA chromosomes, as a guide for more detailed molecular-genetic investigations of the mutations underlying the disorder.     

Four DNA polymorphisms in the LDL-receptor gene and their use in diagnosis of familial hypercholesterolemia

Summary To examine the potential usefulness of restriction fragment length polymorphisms (RFLPs) for diagnosis of familial hypercholesterolemia (FH), we determined the genotype of FH patients and their relatives for the ApalI, NcoI, PvuII and StuI RFLP of the LDL-receptor gene in a sample of German patients attending the Lipid Clinic in Munich. There was no significant difference in the relative allele frequency between the group of FH patients and controls for any of the four polymorphisms. Using linkage analysis, we could determine the four-RFLP haplotypes of 39 defective and 90 normal LDL-receptor genes in 38 FH families. In our sample, defective LDL-receptor genes occur on 6 different chromosomes determined by the four RFLPs. This suggests that at least 6 different genetic defects may cause FH in this sample. RFLPs of the LDL-receptor gene cannot be used to detect FH in individuals; however, appropriate diagnosis can be carried out in more than 90% of families using linkage analysis and these RFLPs.     

Polymorphic DNA haplotypes at the human low-density lipoprotein receptor gene locus in Koreans

Many low-density lipoprotein (LDL) receptor mutations have been identified and characterized, demonstrating a high degree of allelic heterogeneity at this locus. The ability to identify mutant LDL-receptor genes for prenatal diagnosis of familial hypercholesterolemia (FH) or to study the role of the LDL-receptor gene in polygenic hypercholesterolemia requires the use of closely linked restriction fragment lenghth polymorphisms (RFLPs). In the present study nine different RFLPs (TaqI, StuI, HincII, BstEII, AvaII, PvuII, MspIA, MspIB, and NcoI) and a sequence variation at Arg450 were used to clarify the characteristics of the LDL-receptor gene in Koreans. A total of 978 LDL-receptor alleles from 244 members of 43 different pedigrees (15 normal and 28 FH pedigrees) and 245 individuals (187 normal and 58 FH) were analyzed. Frequencies of these polymorphisms did not differ significantly between controls and FH patients. Individually, seven sites--TaqI, BstEII, AvaII, MspIA, MspIB, NcoI and Arg450--had heterozygosity indices ranging from 0.3610 to 0.4601, whereas the PvuII site displayed low levels of polymorphism and StuI was monomorphic. Haplotypes were constructed for 215 individuals of 13 normal and 24 FH pedigrees using the nine polymorphisms. Of 512 (= 2(9)) possible combinations for the nine polymorphic sites, 39 unique haplotypes were detected. The frequency distribution of individual haplotypes ranged from 1/155 (0.65%) to 40/155 (25.8%). The four most common haplotypes accounted for 59.4% of those sampled. Statistical analysis of the haplotypes indicated marked linkage disequilibrium for these 10 sites and throughout the region containing the LDL-receptor gene. Owing to the high degree of linkage disequilibrium over the entire locus, not all RFLPs were informative. We rank each RFLP according to its informativeness and present a strategy for the optimal selection of RFLPs for pedigree analysis.     

Gene arrangement at the Rhesus blood group locus of chimpanzees detected by fiber-FISH

The Rhesus (Rh) blood group system in humans is encoded by two genes with high sequence homology. These two genes, namely, RHCE and RHD, have been implied to be duplicated during evolution. However, the genomic organization of Rh genes in chimpanzees and other nonhuman primates has not been precisely studied. We analyzed the arrangement of the Rh genes of chimpanzees (Pan troglodytes) by two-color fluorescence in situ hybridization on chromatin DNA fibers (fiber-FISH) using two genomic DNA probes that respectively contain introns 3 and 7 of human RH genes. Among the five chimpanzees studied, three were found to be homozygous for the two-Rh-gene type, in an arrangement of Rh (5'-->3') - Rh (3'<--5'). Although a similar gene arrangement can be detected in the RH gene locus of typical Rh-positive humans, the distance between the two genes in chimpanzees was about 50 kb longer than that in humans. The remaining two chimpanzees were homozygous for a four-Rh-gene type, in an arrangement of Rh (5'-->3') - Rh (3'<--5') - Rh (3'<--5') - Rh (3'<--5') within a region spanning about 300 kb. This four-Rh-gene type has not been detected in humans. Further analysis of other great apes showed different gene arrangements: a bonobo was homozygous for the three-Rh-gene type; a gorilla was heterozygous for the one-Rh- and two-Rh-gene types; an orangutan was homozygous for the one-Rh-gene type. Our findings on the intra- and interspecific genomic variations in the Rh gene locus in Hominoids would shed further light on reconstructing the genomic pathways of Rh gene duplication during evolution.     

Gene organization and rearrangements at the human Rhesus blood group locus revealed by fiber-FISH analysis

The human Rhesus (Rh) blood group locus is composed of two highly homologous genes, the RHD and RHCE genes on chromosome 1, encoding the D, C/c, and E/e antigens in common Rh-positive phenotypes. In general, the RHD gene is either absent or grossly deleted in Rh-negative individuals. In this study, gene organization at the RH locus of Japanese donors with different serological phenotypes was directly analyzed by two-color fluorescence in situ hybridization on DNA fibers released from their lymphocytes (fiber-FISH) and by using DNA probes of introns 3 and 7 of the RHCE and RHD genes. Six Rh-positive samples (two with the D+C-c+E+e-, two with the D+C+c-E-e+, and two with the D+C+c+E+e+ phenotype) showed the presence of two RH genes within a region of less than 200 kb on chromosome 1p36.1. Of great interest was the finding that the genes were arranged in the antidromic order of the telomere -RHCE (5'--> 3') -RHD (3'-->5') - centromere. On the other hand, two typical Rh-negative samples (D-C-c+E+e+) showed the presence of only one RHCE gene, as expected. Moreover, further analysis combined with a locus-specific assay of three Rh-negative samples (D-C+c+E+e+, D-C+c+E-e+, and D-C+c-E-e+) showed the possible presence of the RHD gene(s) and complex rearrangements, including partial deletion, duplication, and recombination, in this region; these could be responsible for the Rh-negative phenotype.     

Immunopurification of the blood group RhD protein from human erythrocyte membranes

Rh proteins are membrane proteins encoded by genes at the blood group RH locus. They are of paramount importance in transfusion medicine, but their function is still unknown. Biochemical and biophysical studies of these proteins are scarce since only minute amounts of the very hydrophobic Rh proteins, can be purified from human erythrocytes. Recently, a human monoclonal antibody (LOR-15C9) was described as having the unique property to recognize the Rh30 protein carrying the major blood group D specificity (RhD protein), either in a membrane detergent extract or when blotted on a membrane. In this report, we describe one-step purification of the RhD protein from detergent extracts of red cell membranes, based on immunoaffinity chromatography carried out with immobilized LOR-15C9 IgG. The technique yielded RhD protein with high purity which was devoid of other associated proteins (RhAG, CD47, LW and GPB) that comprise the Rh complex in the erythrocyte membrane. By contrast immunoprecipitation performed with the same antibody led to co-isolation of both RhD and RhAG.     

Haplotypes at ATM identify coding-sequence variation and indicate a region of extensive linkage disequilibrium

Genetic variation in the human population may lead to functional variants of genes that contribute to risk for common chronic diseases such as cancer. In an effort to detect such possible predisposing variants, we constructed haplotypes for a candidate gene and tested their efficacy in association studies. We developed haplotypes consisting of 14 biallelic neutral-sequence variants that span 142 kb of the ATM locus. ATM is the gene responsible for the autosomal recessive disease ataxia-telangiectasia (AT). These ATM noncoding single-nucleotide polymorphisms (SNPs) were genotyped in nine CEPH families (89 individuals) and in 260 DNA samples from four different ethnic origins. Analysis of these data with an expectation-maximization algorithm revealed 22 haplotypes at this locus, with three major haplotypes having frequencies > or = .10. Tests for recombination and linkage disequilibrium (LD) show reduced recombination and extensive LD at the ATM locus, in all four ethnic groups studied. The most striking example was found in the study population of European ancestry, in which no evidence for recombination could be discerned. The potential of ATM haplotypes for detection of genetic variants through association studies was tested by analysis of 84 individuals carrying one of three ATM coding SNPs. Each coding SNP was detected by association with an ATM haplotype. We demonstrate that association studies with haplotypes for candidate genes have significant potential for the detection of genetic backgrounds that contribute to disease.     

A genetic linkage map of 27 loci from PND to FY on the short arm of human chromosome I.

A genetic linkage map of 27 loci on the short arm of human chromosome 1 has been developed by analysis of the 40 families in the Centre d'Etude du Polymorphisme Humain (CEPH) reference panel. Probes that recognize 14 novel RFLPs at loci designated D1S9-D1S22 were isolated from a flow-sorted chromosome 1 library. A linkage map of chromosome 1p was constructed from the genotypic data at these 14 loci, RFLPs at eight cloned genes (PND, ALPL, FUCA1, SRC2, MYCL, GLUT, TSHB, and NGFB), two previously identified RFLPs (D1S2 and D1S57), two blood group antigens (RH and FY), and the isozyme PGM1. All 27 loci form a continuous linkage group, from FY to PND, of 102 cM in males and 230 cM in females. This map provides a basis for highly informative multipoint mapping studies for most of the short arm of chromosome 1.     

Cleft lip with or without cleft palate: associations with transforming growth factor alpha and retinoic acid receptor loci.

The first association study of cleft lip with or without cleft palate (CL/P), with candidate genes, found an association with the transforming growth-factor alpha (TGFA) locus. This finding has since been replicated, in whole or in part, in three independent studies. Here we extend our original analysis of the TGFA TaqI RFLP to two other TGFA RFLPs and seven other RFLPs at five candidate genes in 117 nonsyndromic cases of CL/P and 113 controls. The other candidate genes were the retinoic acid receptor (RARA), the bcl-2 oncogene, and the homeobox genes 2F, 2G, and EN2. Significant associations with the TGFA TaqI and BamHI RFLPs were confirmed, although associations of clefting with previously reported haplotypes did not reach significance. Of particular interest, in view of the known teratogenic role of retinoic acid, was a significant association with the RARA PstI RFLP (P = .016; not corrected for multiple testing). The effect on risk of the A2 allele appears to be additive, and although the A2A2 homozygote only has an odds ratio of about 2 and recurrence risk to first-degree relatives (lambda 1) of 1.06, because it is so common it may account for as much as a third of the attributable risk of clefting. There is no evidence of interaction between the TGFA and RARA polymorphisms on risk, and jointly they appear to account for almost half the attributable risk of clefting.